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. 2021 Jul 26;95(16):e0061721.
doi: 10.1128/JVI.00617-21. Epub 2021 Jul 26.

V367F Mutation in SARS-CoV-2 Spike RBD Emerging during the Early Transmission Phase Enhances Viral Infectivity through Increased Human ACE2 Receptor Binding Affinity

Junxian Ou #  1 , Zhonghua Zhou #  2 , Ruixue Dai  3 , Jing Zhang  4 , Shan Zhao  1 , Xiaowei Wu  1 , Wendong Lan  1 , Yi Ren  4 , Lilian Cui  5 , Qiaoshuai Lan  6 , Lu Lu  6 , Donald Seto  7 , James Chodosh  8 , Jianguo Wu  4 , Gong Zhang  2 , Qiwei Zhang  1   4
Affiliations

V367F Mutation in SARS-CoV-2 Spike RBD Emerging during the Early Transmission Phase Enhances Viral Infectivity through Increased Human ACE2 Receptor Binding Affinity

Junxian Ou et al. J Virol. .

Abstract

The current pandemic of COVID-19 is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 spike protein receptor-binding domain (RBD) is the critical determinant of viral tropism and infectivity. To investigate whether naturally occurring RBD mutations during the early transmission phase have altered the receptor binding affinity and infectivity, we first analyzed in silico the binding dynamics between SARS-CoV-2 RBD mutants and the human angiotensin-converting enzyme 2 (ACE2) receptor. Among 32,123 genomes of SARS-CoV-2 isolates (December 2019 through March 2020), 302 nonsynonymous RBD mutants were identified and clustered into 96 mutant types. The six dominant mutations were analyzed applying molecular dynamics simulations (MDS). The mutant type V367F continuously circulating worldwide displayed higher binding affinity to human ACE2 due to the enhanced structural stabilization of the RBD beta-sheet scaffold. The MDS also indicated that it would be difficult for bat SARS-like CoV to infect humans. However, the pangolin CoV is potentially infectious to humans. The increased infectivity of V367 mutants was further validated by performing receptor-ligand binding enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, and pseudotyped virus assays. Phylogenetic analysis of the genomes of V367F mutants showed that during the early transmission phase, most V367F mutants clustered more closely with the SARS-CoV-2 prototype strain than the dual-mutation variants (V367F+D614G), which may derivate from recombination. The analysis of critical RBD mutations provides further insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin under negative selection pressure and supports the continuing surveillance of spike mutations to aid in the development of new COVID-19 drugs and vaccines. IMPORTANCE A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the pandemic of COVID-19. The origin of SARS-CoV-2 was associated with zoonotic infections. The spike protein receptor-binding domain (RBD) is identified as the critical determinant of viral tropism and infectivity. Thus, whether mutations in the RBD of the circulating SARS-CoV-2 isolates have altered the receptor binding affinity and made them more infectious has been the research hot spot. Given that SARS-CoV-2 is a novel coronavirus, the significance of our research is in identifying and validating the RBD mutant types emerging during the early transmission phase and increasing human angiotensin-converting enzyme 2 (ACE2) receptor binding affinity and infectivity. Our study provides insights into the evolutionary trajectory of early SARS-CoV-2 variants of zoonotic origin. The continuing surveillance of RBD mutations with increased human ACE2 affinity in human or other animals is critical to the development of new COVID-19 drugs and vaccines against these variants during the sustained COVID-19 pandemic.

Keywords: ACE2 receptor; COVID-19; SARS-CoV-2; mutation; receptor-binding domain (RBD); variants; viral infectivity.

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Figures

FIG 1
FIG 1
Geographical distribution of the SARS-CoV-2 RBD mutants. The geographic distribution of the RBD mutants on four continents (>10 isolates) is displayed. The mutants marked in black are mutants with similar binding affinities as strain Wuhan-Hu-1. V367F mutants with the enhanced binding affinity were found on all of the four continents and are marked in red. The mutants analyzed were isolated as of 31 March 2020.
FIG 2
FIG 2
Mutation scanning graphs of the SARS-CoV-2 S gene. Mutation sites compared to strain Wuhan-Hu-1 were analyzed by BioAider (V1.314) (29). (A) Total mutations (synonymous and nonsynonymous) in the spike gene. (B) Nonsynonymous and synonymous mutations in the spike gene. (C) Total mutations and nonsynonymous mutations in the RBD. All positions containing gaps and missing data were eliminated. Structural domains are annotated. The peak signals of D614G and the other dominant mutations are marked in red. The ordinates show the numbers of mutants. The isolates analyzed were isolated as of 31 March 2020.
FIG 3
FIG 3
Binding free energy calculated for the SARS-CoV-2 S-RBD to human ACE2 receptor. (A) RMSD of typical MD trajectories of the SARS-CoV-2 prototype and the mutants. (B) Comparison of the binding free energy (ΔG) of the RBDs of human SARS-CoV-2, bat CoV, and pangolin CoV to the human ACE2. Note the ΔG is inversely proportional to the binding affinity. Data are presented as means ± standard deviations (SDs). P values were calculated using single-tailed Student's t test. The P values are shown for those with a P value of <0.05. The ΔG calculated from experimental KD values of SARS and the SARS-CoV-2 prototype are marked in dotted and dashed lines, respectively. (C) Comparison of the equilibrium dissociation constants (KD) calculated with the ΔG.
FIG 4
FIG 4
Structural analysis of RBD mutants and the effects on their binding affinity. (A) Binding surface and interaction of the RBD to ACE2, with the locations of the mutant amino acids noted. Beta-sheet structure scaffold was centered by residues 510 to 524 (in red). (B) Root mean square of fluctuation (RMSF) values of the mutants were compared to that of the prototype. Red arrows denote the fragment of residues 510 to 524. Black arrows denote the fragment of residues 475 to 485. (C) Contribution of each amino acid to the binding free energy. Red bars denote the binding site.
FIG 5
FIG 5
Experimental validation of the enhanced affinity and infectivity of the V367F mutant. (A) Comparison of the binding affinity of prototype S protein and V367F mutant to human ACE2 receptor by ligand-receptor binding ELISA. (B) Comparison of the binding affinity of prototype S protein and V367F mutant to human ACE2 protein by SPR. (C) Quantification of the genome copy number of the V367F mutant versus the prototype using pseudovirus infection assay. The relative fold increases of viruses infecting the cells are shown by the pseudoviral DNA copy number of the V367F mutant in both Vero and Caco-2 cells at 24 h p.i. and 48 h p.i. Experiments were performed in triplicates, and the P values were calculated using two-tailed t test for two samples with different variances.
FIG 6
FIG 6
Whole-genome phylogenetic analysis of the SARS-CoV-2 variants emerging during the early transmission phase (December 2019 through March 2020). The whole-genome phylogenetic tree was constructed by IQ-Tree 2.02 using the maximum likelihood method with GTR+F+R3 model, 1,000 bootstrap replicates, and applying default parameters. All 34 V367F mutants were included. For reference, the branch of Wuhan-Hu-01 is marked in black. V367F mutants are marked with red triangles; sampled referenced sequences are annotated in different colors by clades using Figtree 1.4.4 (https://github.com/rambaut/figtree/releases).
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